This application is related to U.S. patent application Ser. No. 08/734,607, filed Oct. 18, 1996, now U.S. Pat. No. 6,210,913; U.S. Provisional Application No. 60/031,665, filed Nov. 21, 1996; U.S. Provisional Application No. 60/042,093, filed Mar. 28, 1997; and, U.S. patent application Ser. No. 08/975,653, filed Nov. 21, 1997. All of the publications and patent applications that are identified in this specification are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
A. Integrins
Integrins are a family of αβ heterodimers that mediate adhesion of cells to extracellular matrix proteins and to other cells (Clark et al., Science (1995) 268:233–239). Integrins also bind to the actin cytoskeleton through a series of intermediate proteins, and thus provide a link between the extracellular matrix and the intracellular cytoskeleton and its associated motile machinery. Such transmembrane linkages are required for cell migration. Many biological responses are dependent at least to some extent upon integrin-mediated adhesion and cell migration, including embryonic development, hemostasis, clot retraction, mitosis, angiogenesis, inflammation, immune response, leukocyte horning and activation, phagocytosis, bone resorption, tumor growth and metastasis, atherosclerosis, restenosis and wound healing.
Members of the integrin family also participate in signal transduction. This is evidenced by an alteration in the adhesive affinity of cell surface integrins in response to cellular activation, termed inside-out signal transduction. Additionally, effects on intracellular signaling pathways following integrin-mediated adhesion have been observed, termed outside-in signal transduction.
The integrin family consists of 15 related known α subunits (α1, α2, α3, α4, α5, α6, α7, α8, α9, αE, αV, αIIb, αL, αM, and αX) and 8 related known β subunits (β1, β2, β3, β4, β5, β6, β7, and β8). Luscinskas et al., FASEB J., 8: 929–938 (1994). Integrin α and β subunits are known to exist in a variety of pairings as indicated in FIG. 1. Integrin ligand specificity is determined by the specific pairing of the α and β subunits, although some redundancy exists as several of the integrins are known to bind the same ligand.
Two known parings of the β3 subunit have been observed: with αV to make αVβ3, the Vitronectin Receptor; and with GP IIb to make GP IIb–IIIa, the Fibrinogen Receptor. αVβ3 is widely distributed, is the most promiscuous member of the integrin family and mediates cellular attachment to a wide spectrum of adhesive proteins, mostly at the R-G-D sequence on the adhesive protein. The biological processes mediated by αVβ3 are diverse and include bone resorption, angiogenesis, tumor metastasis and restenosis. αVβ3 is known to signal upon adhesive protein ligation (P. I. Leavesley, et al., J. Cell Biol. 121:163–170 (1993)). As an example, endothelial cells undergo apoptosis when relieved of ligation (P. C. Brooks, Cell 79:1157–1164 (1994)).
B. Interaction of Integrins with Known Cytoskeletal Proteins
The binding of unmodified α and β subunit cytoplasmic domains of integrins to a variety of cytoskeletal and signaling proteins has been documented. S. Dedhar et al., Curr. Opin. Cell Biol. 8:657–669 (1996). Morphological studies have shown that many of these proteins are concentrated in focal adhesions where integrins cluster and bind to both the extracellular matrix and cytoskeletal proteins. I. Knezevic et al., J. Biol. Chem. 271(27):16416–16521 (1996).
For example, talin, a 235 kD vinculin and actin binding protein, binds to the cytoplasmic domains of αIIb and β3 in a solid phase binding assay. I. Knezevic et al., Id. The binding of α actinin, a 100 kD vinculin binding protein and actin cross-linking protein, to the cytoplasmic domain of β1 and β3 in solid phase binding assays has also been observed. C. A. Otey et al., J. Biol. Chem. 268(28); 21193–21197 (1993); and C. A. Otey et al., J. Cell Biol. 111:721–729 (1990). Binding studies have demonstrated an interaction between the cytoplasmic domain of β1 and tensin, a 215 kD SH2 domain containing vinculin and actin binding protein. S. Lin et al. Mol. Biol. Cell 7 Supp. 389a, Abstract 2259 (1996).
Other cytoskeletal related proteins also interact with integrins. Skelemin, a 195 kD myosin and intermediate filament binding protein, binds to the membrane proximal regions of β1 and β3 cytoplasmic domains. K. B. Reddy et al., Mol. Biol. Cell 7 Supp. 385A, Abstract 2237 (1995). These authors suggested that skelemin could link myosin and intermediate filaments to β integrins.
Paxillin, a vinculin binding signaling protein also binds to the cytoplasmic domain of the β1 integrin. M. D. Schaller et al., J. Cell Biol. 130:1181–1187 (1995). It is not yet known whether the β1-paxillin association is direct or indirect, however paxillin was postulated as being the substrate for and tyrosine phosphorylated by tyrosine kinase pp125 FAK. The actin binding protein filamin has been shown to bind to the cytoplasmic tail of the β2 integrin subunit in vitro and co-immunoprecipitated and co-localized with β2 integrins in vivo. C. P. Sharma et al., J. Immunol. 154; 3461–3470 (1995).
A 208 kD integrin binding protein identified as being related to the myosin light chain kinase family of serine/threonine kinases has also been reported. Walker et al., Mol. Biol. Cell 7 Supp. 385A, Abstract 2235 (1995). This kinase was said to be part of a complex of proteins including α-actinin and myosin, however, it was unclear whether the kinase associated directly with the cytoplasmic tails of integrins or through a complex of proteins.
Although the cytoskeletal proteins listed above have been shown to interact with cytoplasmic domains of integrin subunits with purified proteins or peptides, it is not known how these interactions occur within cells or how these interactions are regulated. Furthermore, the integrin/cytoskeletal interactions described thus far do not occur in a phosphotyrosine-dependent manner.
C. Tyrosine Phosphorylation of the Cytoplasmic Domain of Integrin β Subunits
Platelet aggregation induced by a number of agonists results in the phosphorylation of tyrosine residues in the β3 cytoplasmic tail. Law et al., J. Biol. Chem 271:10811–10815 (1996). In some respects, the phosphorylation of both tyrosine residues was necessary for binding to certain signaling proteins, whereas other signaling proteins bound following monophosphorylation. Furthermore, adhesion to vitronectin by cells transfected with αvβ3 induces a robust tyrosine phosphorylation of the β3 subunit. Blystone et al., J. Biol. Chem 271:31458–31462 (1996).
Studies have shown that the sequences of the cytoplasmic domains of β1, β2 and β3 which contain tyrosines are important for normal integrin/cytoskeletal interactions. For example, the substitution of tyrosine 747 by alanine in β3 transfected into CHO cells abolished β3-mediated cell spreading, blocked the recruitment of αIIbβ3 to preestablished adhesion plaques, and decreased the ability of αIIbβ3 to mediate internalization of fibrinogen-coated particles. J. Ylanne et. al., J. Biol. Chem., 270, 9550–9557, (1995).
Additional experiments reported by Ylanne et al., Id., showed further that substitution of alanine for tyrosine 759 decreased cell spreading and the recruitment of αIIbβ3 to adhesion plaques, while deletion of the carboxy terminal pentapeptide that contains this sequence had an even more pronounced effect on the function of the integrin. These authors concluded integrin-mediated cell spreading does not occur because the factors that are absolutely required for integrin-mediated cell spreading cannot bind either the β3 truncated at residue 757 or the integrin with tyrosine 747 of β3 substituted by alanine.
Point mutations in homologous domains in β1- and β2-containing integrins also modulate function, as these mutations affect integrin-cytoskeletal interactions by reducting focal adhesions, A. A. Reszka et. al., J. Cell Biol. 117:1321–1330 (1992), and integrin activation, M. L. Hibbs et. al., J. Exp. Med. 174:1227–1238 (1991), respectively. Tyrosine kinases similarly were found to be essential in regulating the cytoskeletal attachment of αIIbβ3. Schoenwaelder et al., J. Biol. Chem. 269(51):32479–32487 (1994).
Overall, the interactions between the two tyrosines and the “cell adhesion regulatory domain” or “CARD” of residues 747–762 of the β3 cytoplasmic domain were reported to be essential for regulation of the adhesive function of integrin β3. Liu et al., PNAS 93:11819–11824 (1996). A 16-amino acid sequence from the CARD inhibited adhesion of HEL and ECV 304 cells to immobilized fibrinogen by competing with intracellular protein-protein interactions that “engage the business end” of the integrin β3 tail. However, the identity of cytoplasmic protein(s) interacting with CARD was said to remain to be established.
D. Myosin
The platelet plasma membrane is coated by a lattice-like structure, known as the membrane skeleton, that is composed of short actin filaments, actin-binding protein, spectrin, vinculin and various other proteins, not all yet identified. Fox et al., J. Biol. Chem. 268(34):25973–25984 (1993). On the cytoplasmic side, the skeleton appears to be associated with a network of cytoplasmic actin filaments. The membrane skeleton coats the lipid bilayer and is associated with both extracellular glycoproteins and intracellular cytoskeletal elements. Fox et al. suggested that GPIIb–IIIa induces redistribution of components of the membrane skeleton and associated signaling molecules as a step in regulating integrin-induced motile events in platelets.
Myosin is a contractile protein that interacts with actin to produce contraction or movement. The term “myosin” broadly refers to a diverse superfamily, comprised of at least 11 classes, of molecular motors capable of translocating actin filaments or of translocating vesicles or other cargo on fixed actin filaments by. One characteristic of all myosins is their ability to reversibly bind to actin and to hydrolyze MgATP. See FIG. 5 and J. R. Sellers and H. V. Goodson, Protein Profile 2:1323–1339 (1995).
All types of myosin that have been purified are multimeric and appear to possess at least three functional domains—a head, neck and tail. The head or motor domain contains nucleotide and actin binding sites and is the most conserved region of the myosin superfamily. The neck domain consists of a long single alpha helical strand from the heavy chain which is stabilized by the binding of light chain subunits. The tail region, which serves to anchor myosin so that it can translocate actin, is the most diverse primary sequence of all the regions and may serve to anchor certain myosin isoforms to cell or organelle membranes. It has been suggested that myosin clustering within a cell may occur on membranes or on actin filaments themselves. Titus, Trends in Cell Biology 7:119 (1997). However, the precise biochemical mechanism of interaction between the myosin tail and cytocellular structures has not heretofore been described.
E. Signal Transduction
The involvement of the cytoplasmic domain of GP IIb–IIIa in integrin signal transduction is inferred from mutagenesis experiments. Deletion of the cytoplasmic domain of GP IIb results in a constitutively active receptor that binds fibrinogen with an affinity equivalent to the wild-type complex, implying that the cytoplasmic tail of GP IIb has a regulatory role (T. E. O'Toole, et al., Cell Regul. 1:883–893, (1990)). Point mutations, deletions and other truncations of GP IIb–IIIa affects the ligand binding activity of GP IIb–IIIa and its signaling response (P. E. Hughes, et al., J. Biol. Chem. 270:12411–12417, (1995); J. Ylanne, et al., J. Biol. Chem. 270:9550–9557, (1995)).
Chimeric, transmembrane proteins containing the cytoplasmic domain of GP IIIa, but not of GP IIb, inhibit the function of GP IIb–IIIa (Y. P. Chen et al., J. Cell Biol. 269:18307–18310, (1994)), implying that free GP IIIa cytoplasmic domains bind proteins within cells which are necessary for normal GP IIb–IIIa function. Several proteins have been shown to bind either the transmembrane domains or the cytoplasmic domains of GP IIb or GP IIIa.
CD-9, a member of the tetraspanin family of proteins (F. Lanza, et al., J. Biol. Chem. 266:10638–10645, 1991), has been found to interact with GP IIb–IIIa on aggregated platelets. β3-endonexin, a protein identified through two hybrid screening using the cytoplasmic domain of GP IIIa as the “bait”, has been found to interact directly and selectively with the cytoplasmic tail of GP IIIa (S. Shattil et al., J. Cell. Biol. 131:807–816, (1995)). β3-endonexin shows decreased binding to the GP IIIa cytoplasmic domain containing the thrombasthenic S752-P mutation. It is not yet known whether either of these GP IIIa-binding proteins are involved in signal transduction.
Cytoplasmic proteins that bind to αVβ3 have also been described which may be interacting with the integrin at the GP IIIa cytoplasmic domain sequence. Bartfeld and coworkers (N. S. Bartfeld et al., J. Biol. Chem. 268:17270–17276, (1993)) used immunoprecipitation from detergent lysates to show that a MW=190-kDa protein associates with the αVβ3 integrin from PDGF-stimulated 3T3 cells. IRS-1 was found to bind to the αVβ3 integrin following insulin stimulation of Rat-1 cells stably transfected with DNA encoding the human insulin receptor (K. Vuori and E. Ruoslahti, Sci. 266:1576–1578, (1994)). Kolanus et al. (Cell 86:233–242, (1996)) recently identified Cytohesin-1. Cytohesin-1 specifically binds to the intracellular portion of the integrin β2 chain, and overexpression of cytohesin-1 induces β2 integrin-dependent binding of Jurkat cells to ICAM-1. A novel serine/threonine kinase, ILK-1, was found to associate with the β1 cytoplasmic domain (Hannigan et al., Nature 379:91–96, (1996)). Overexpression of ILK-1 inhibits adhesion to the integrin ligands fibronectin, laminin, and vitronectin.
Integrin binding to adhesive proteins and integrin signal transduction have a wide variety of physiological roles, as identified above. Enhanced signaling through integrins allows for increased cell adhesion and activation of intracellular signaling molecules which causes enhanced cell mobility and growth, enhanced cell responsiveness, and modulations in morphological transformations. Although integrins responsible for cellular function have been described and signaling events are beginning to be elucidated, the mechanism by which integrins transduce signals remains to be determined. To understand the molecular mechanisms of the inside-out and outside-in signaling roles mediated by the cytoplasmic tails of β3 integrin requires the identification of the intracellular molecules that interact with the intracellular tails of integrin. It has been reported that α-actinin binds to β1 tails in vitro (Otey et al. J. Biol. Chem. 268:21193–21197, (1993)) but the functional relevance of these bindings is not clear. By using yeast two-hybrid, ILK-1 was identified as a β1 interacting protein but ILK-1 does not bind to β3 (Hannigan et al., Nature 379:91–96 (1996)).
F. Homologous Recombination
Genes can be introduced in a site directed fashion using homologous recombination. This can be used in the creation of a transgenic animal, wherein the animal would be mutated, and the phenotype of the mutation could be studied for purposes of drug screening, investigating physiologic processes, developing new products and the like. Papers discussing homologous recombination are discussed in R. Kucherlapati et al., (1995) U.S. Pat. No. 5,413,923.
Homologous recombination permits site-specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome. The application of homologous recombination to gene therapy depends on the ability to carry out homologous recombination or gene targeting in normal, somatic cells for transplantation.
To prepare cells for homologous recombination, embryonic stem cells or a stem cell line may be obtained. Cells other than embryonic stem cells can be utilized (e.g. hematopoietic stem cells etc.) (See for more examples, J. G. Seidman et al., (1994) U.S. Pat. No. 5,589,369). The cells may be grown on an appropriate fibroblast fetal layer or grown in the presence of leukemia inhibiting factor (LIF) and then used. The embryonic stem cells may be injected into a blastocyst, that has been previously obtained, to provide a chimeric animal. The main advantage of the embryonic stem cell technique is that the cells transfected with the “transgene” can be tested prior to reimplantation into a female animal for gestation for integration and the effect of the transgenes. In contrast to the conventional microinjection technique, the homologous respective endogenous gene can be removed from a chromosome by homologous recombination with the transgene. By subsequent cross-breeding experiments, animals can be bred which carry the transgene on both chromosomes. If mutations are incorporated into the transgenes which block expression of the normal gene production, the endogenous genes can be eliminated by this technique and functional studies can thus be performed.
Homologous recombination can also proceed extrachromasomally, which may be of benefit when handling large gene sequences (e.g., larger than 50 kb). Methods of performing extrachromosomal homologous recombination are described in R. M Kay et al., (1998) U.S. Pat. No. 5,721,367.
G. Production of Transgenic Animals
Transgenic animals are genetically modified animals into which cloned genetic material has been experimentally transferred. The cloned genetic material is often referred to as a transgene. The nucleic acid sequence of the transgene is integrated at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found. The transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.
The development of transgenic technology allows investigators to create mammals of virtually any genotype and to assess the consequences of introducing specific foreign nucleic acid sequences on the physiological and morphological characteristics of the transformed animals. The availability of transgenic animals permits cellular processes to be influenced and examined in a systematic and specific manner not achievable with most other test systems. For example, the development of transgenic animals provides biological and medical scientists with models that are useful in the study of disease. Such animals are also useful for the testing and development of new pharmaceutically active substances.
Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,602,307; Mullins et al., Hypertension 22(4):630–633 (1993); Brenin et al., Surg. Oncol. 6(2)99–110 (1997); Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1997)). The term “knock-out” generally refers to mutant organisms, usually mice, which contain a null allele of a specific gene. The term “knock-in” generally refers to mutant organisms, also usually mice, into which a gene has been inserted through homologous recombination. The knock-in gene may be a mutant form of a gene which replaces the endogenous, wild-type gene.
A number of recombinant murines have been produced, including those which express an activated oncogene sequence (U.S. Pat. No. 4,736,866); express simian SV 40 T-antigen (U.S. Pat. No. 5,728,915); lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Pat. No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Pat. No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Pat. No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Pat. No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S. Pat. No. 5,602,307); possess an bovine growth hormone gene (Clutter et al., Genetics 143(4):1753–1760 (1996)); and, are capable of generating a fully human antibody response (McCarthy, The Lancet 349(9049):405 (1997)).
While murines, especially mice and rats, remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species. Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al., Mol. Reprod. Dev. 46(4(:515–526 (1997); Houdebine, Reprod. Nutr. Dev. 35(6):609–617 (1995); Petters, Reprod. Fertil. Dev. 6(5):643–645 (1994); Schnieke et al., Science 278(5346):2130–2133 (1997); and, Amoah, J. Animal Science 75(2):578–585 (1997)).
The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the recitations in U.S. Pat. No. 5,489,743 and U.S. Pat. No. 5,602,307.